Method of screening cell growth inhibitor and cell growth inhibitor

ABSTRACT

The present invention provides a method for screening a cytostatic agent characterized by determining an effect of inhibiting intracellular signaling mediated by a remaining fragment of a cell membrane-anchored growth factor, and the cytostatic agent. In growth factors of the EGF family, the physiological significance of the remaining fragment is clarified, and a method for controlling its action is searched, and thus a novel pharmaceutical can be provided.

TECHNICAL FIELD

The present invention relates to a method for screening a cytostaticagent capable of inhibiting an intracellular signaling process that hasnot been conventionally known. The method of the present invention issignificantly useful as a method for screening cytostatic agents, inparticular, antitumor agents, antirheumatic agents, agents for treatingcardiac disorder, etc. that are based on a novel action mechanism.

BACKGROUND ART

A large number of cell membrane-anchored growth factors that aresynthesized in vivo as a cell membrane-anchored precursor, becomesoluble in response to cleavage by a processing enzyme (shedding) andexpress their actions are known.

For example, a heparin-binding EGF-like growth factor (HB-EGF),amphiregulin, epiregulin, transforming growth factor-α (TGF-α) and thelike are members of the epidermal growth factor (EGF) family, and it hasbeen clarified that all of the growth factors that belong to the EGFfamily are biosynthesized as a cell membrane-anchored precursor andbecome soluble in response to processing. It is known that in general,the solubilized growth factor is then bound to a receptor of the ErbBreceptor family in an activated form so as to express the action of agrowth factor.

It is known that similarly to these growth factors of the EGF family,tumor necrosis factors (TNF-α) express a large number of physiologicalactions in an activated form after the cell membrane-anchored precursorhas been subjected to processing by proteases.

However, in any case, the role of a fragment that is left in the cellsurface (hereinafter, referred to as “remaining fragment”) after theactivated form is released by processing has not been known.

On the other hand, the mechanism in which a membrane-anchored receptorreleases a signal transducer (second messenger) in response toextracellular stimulations and transduces external signals to a cell iswell known. In recent years, a signaling mechanism (regulatedintramembrane proteolysis; RIP) has been presented and gained muchattention, in which processing of an extracellular domain of amembrane-anchored protein causes a remaining fragment to be translocatedinto the nucleus, after being subjected to further processing in somecases, and to regulate the transcription of a gene (M. S. Brown et al.,Cell, 100, 391-396(2000)).

However, in this as well, only the fact of signaling into the nucleushas been identified, but the meaning of the signal, and thephysiological significance such as the subsequent phenomenon caused bythe transduced signals have not been clarified yet.

Furthermore, at least regarding HB-EGF, the behavior itself of theremaining fragment after processing has not been known at all

Cell, 100, 391-396 (2000) (M. S. Brown et al.)

DISCLOSURE OF INVENTION

In the growth factors of the EGF family including HB-EGF, thephysiological significance of the remaining fragment is not known atall. It is an object of the present invention to identify itsphysiological significance and to provide a novel method for regulatingits action, and a novel pharmaceutical.

Among a variety of membrane-anchored growth factors, the inventors ofthe present invention focus particularly on HB-EGF and transfectedHB-EGF whose carboxy-terminal (C terminal) in the intracellular domainis modified with a fluorescence into a cell for the purpose of tracingthe remaining fragment. Then, the manner in which after theextracellular domain of the HB-EGF is subjected to processing by asolubilizing enzyme, the tagged intracellular domains accumulate aroundthe nucleus was observed under a microscope.

Since the transport of the remaining fragment into the nucleus wassuggested, then an intranuclear protein that interacts with theremaining fragment of the HB-EGF was searched. As a result of searchinga human heart cDNA library with a system employing a yeast (two-hybridsystem; Clontech), using the intracellular domain (185-208aa) of theHB-EGF as bait, promyelocytic leukemia zinc finger (hereinafter,referred to as PLZF) protein was found out as a protein that interactstherewith.

It is known that the PLZF protein suppresses the cell growth bysuppressing the action of cyclin A in the cell nucleus (Yeyati et al.,Oncogene, 18, 925-934(1999)). Therefore, it was suggested that theremaining fragment of the HB-EGF might participate in the control ofcell growth by interacting with the PLZF protein.

As a result of in-depth research, the inventors of the present inventionfound out the following facts.

1) In the cells in which a tagged PLZF protein is transfected into itsnucleus, when the extracellular domain of the HB-EGF is subjected toprocessing by a solubilizing enzyme, the remaining fragment is bound toa region including C₂H₂Zn finger motifs of the tagged PLZF protein.2) The nuclear export of the tagged PLZF protein is observed.3) Cyclin A is expressed in the nucleus in response to the nuclearexport of the tagged PLZF protein.4) With the expression of cyclin A, a reduction in the amount ofinternalized bromodeoxyuridine (BrdU) is observed, and the progressionof the cell cycle is seen.

Then, the fact that the nuclear export described in 2) is inhibited bythe addition of an antitumor agent reptomycin B (see the example below)was confirmed, and thus the present invention has been achieved.

Hereinafter, the present invention will be described in detail.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the nuclear export effect of an intranuclearPLZF protein by processing. The vertical axis shows the ratio (%) of thenumber of cells expressing CFP-PLZF in the nucleus with compared to thetotal number of the CFP-PLZF transfectant cells. The colored bar graphshows the results without TPA treatment, and the colorless bar graphshows the results with TPA treatment. The horizontal axis shows HB1080,HB1080/HB-EGF, HT1080/ΔMP-ADAM12/HB-EGF, and HT1080/HB-EGF-UCrespectively.

FIG. 2 shows graphs representing the effect of the nuclear export of theintranuclear PLZF protein by processing on the expression ofintranuclear cyclin A. The vertical axis shows the fluorescenceintensity of the fluorescence-tagged PLZF protein, and the horizontalaxis shows the fluorescence intensity of cyclin A.

FIG. 3 shows graphs representing the effect of processing on the amountof BrdU internalized through the nuclear export of the intranuclear PLZFprotein and the activation of the intranuclear cyclin A. (±) in TPA inthe horizontal axis shows whether or not TPA treatment is performed, and(±) in PLZF protein shows the tagged PLZF proteintransfectant/non-transfectant, respectively. The vertical axis shows theratio (%) of the number of BrdU-internalized cells compared to thenumber of the transfectant or non-transfectant cells that are subjectedto the test.

FIG. 4 is a graph showing the nuclear export effect of Bcl-6 protein byprocessing. The vertical axis shows the ratio (%) of the number of cellsexpressing CFP-Bcl-6 in the nucleus compared to the total number of theCFP-Bcl-6 transfectant cells. The colored bar graph shows the resultswithout TPA treatment, and the colorless bar graph shows the resultswith TPA treatment. The horizontal axis shows HB1080 and HB1080/HB-EGFrespectively.

FIG. 5 is a graph showing the nuclear export effect of myoneurin proteinby processing. The vertical axis shows the ratio (%) of the number ofcells expressing CFP-myoneurin in the nucleus compared to the totalnumber of the CFP-myoneurin transfectant cells. The colored bar graphshows the results without TPA treatment, and the colorless bar graphshows the results with TPA treatment. The horizontal axis shows HB1080and HB1080/HB-EGF respectively.

FIG. 6 is a graph showing the nuclear export effect of KIAA0441 protein.The vertical axis shows the ratio (%) of the number of cells expressingCFP-KIAA0441 in the nucleus compared to the total number of theCFP-KIAA0441 transfectant cells. The colored bar graph shows the resultswithout TPA treatment, and the colorless bar graph shows the resultswith TPA treatment. The horizontal axis shows HB1080 and HB1080/ERrespectively.

FIG. 7 shows a graph representing the inhibitory effect of reptomycin Bon the nuclear export of the PLZF protein by processing. The verticalaxis shows the ratio (%) of the number of cells expressing CFP-PLZF inthe nucleus compared to the total number of the CFP-PLZF transfectantcells. (±) in TPA in the horizontal axis shows whether or not TPAtreatment is performed, and (±) in LMB shows the presence or the absenceof reptomycin B, which is a compound to be tested, respectively.

BEST MODE FOR CARRYING OUT THE INVENTION

“Screening” referred to in the present invention includes not onlyso-called first screening for selecting the inhibitor of the presentinvention among a plurality of candidates, but also secondary screening(confirmation test) for confirming the antitumor action (cytostaticability) of a test substance.

In the present invention, a method for determining an effect ofinhibiting intracellular signaling substantially contains the steps of,for each of the cases in the presence and the absence of a testsubstance, (1) producing a remaining fragment of a cellmembrane-anchored growth factor by processing of the growth factor, and(2) determining the extent of the nuclear export of a taggedtranscriptional regulator.

The cell membrane-anchored growth factor refers to a growth factor whoseprecursor is present on the surface of a cell membrane (ectodomainregion) and that is known to exert an action by becoming soluble inresponse to cleavage with a processing enzyme (shedding). For example,the cell membrane-anchored growth factor includes the epidermal growthfactor family (EGF family) including epiregulin, amphiregulin,transforming growth factor-α (TGF-α), neuregulin, and NTAK, in additionto HB-EGF.

In the present specification, the remaining fragment includes thefollowing (X), (Y) and (Z).

-   (X) Fragment that remains after the activated form is released by    processing.-   (Y) Fragment in which at least a portion of the domain inside the    cell membrane of (X) is cleaved by further processing.-   (Z) Proteins having the same amino acid sequence as that of (X) or    (Y), regardless of the production process.

(Y) tends to be easily freed from a cell membrane by further processing.

The transcriptional regulator refers to a protein (repressor) thatinhibits the action of RNA polymerase by binding to a promoter portionof DNA so as to regulate gene expression, and receives signaling by theremaining fragment of the cell membrane-anchored growth factor. Thetranscriptional regulator includes histone deacetylase (HDAC) such asHDAC5, HDAC7, and HDAC9 and splicing isoform thereof, proteins havingBTB/C₂H₂Zn finger motifs, and the like. Specific examples of theproteins having BTB/C₂H₂Zn finger motifs include PLZF proteins, Bcl-6proteins, myoneurin proteins, and KIAA0441 (“Kazusa DNA ResearchInstitute” code number) proteins.

Any cell can be used as a test cell, as long as it expresses a growthfactor targeted by processing. However, in order to analyze the effectof a specific remaining fragment on signaling, it is preferable tosimplify a screening system and use a cell that has expressed only aspecific growth factor. For example, in the case of HB-EGF, humanfibrosarcoma HT1080 cells to which HB-EGF has been transfected(H1080/HB-EGF) can be used preferably.

In the present invention, not only a wild-type HB-EGF, but also HB-EGFwith partial deletion, substitution or addition of the amino acids thatdoes not impair the interaction activity with the intranucleartranscriptional regulator can be used.

For example, the inventors of the present invention confirmed that evenif HB-EGF with deletion of 10 amino acids in the C-terminal is used,remaining fragments accumulate around the nucleus, and that in responseto processing, the nuclear export of the intranuclear PLZF proteinoccurs. The inventors of the present invention further confirmed thatthe substitution of amino acids having charges in the remaining fragmentprevents the nuclear exports.

This suggests that the main active portion of the HB-LGF for the nuclearexport of the PLZF protein is present in a portion of the amino acidsequence from the 185^(th) to 198 ^(th) amino acids (SEQ. ID. NO:4) andthat the presence of amino acid residues having positive charges ornegative charges is essential for the nuclear export.

Therefore, deleted or substituted-type HB-EGF in which the nuclearexport of the PLZF protein can occur is the following (1) or (2), forexample.

-   (1) Deleted-type HB-EGF in which at least one of the amino acids    corresponding to an amino acid sequence portion with 10 amino acids    from the 199^(th) to amino acids (SEQ. ID. NO:5) in the so-called    cyto region (cytoplasmic domain), that is, an amino acid sequence    portion with 24 amino acids from the 185^(th) to 208^(th) amino    acids (more specifically, RYHRRGGYDVENEEKVKLGMTNSH (SEQ. ID. NO:1)).-   (2) Substituted-type HB-EGF in which in the portion after the    185^(th) amino acid of the amino acid sequence of wild-type HB-EGF    or HB-EGF described in (1), a non-charged amino acid residue is    substituted with another non-charged amino acid residue.

Therefore, a method for producing a cytostatic agent of the presentinvention can be achieved by screening a substance that is bound to anamino acid sequence portion with 14 amino acids of the 185^(th) to198^(th) amino acids (SEQ. ID. NO:4) from the amino terminal of aheparin-binding LGF-like growth factor.

In the screening method of the present invention, when performingprocessing a cell membrane-anchored growth factor, it is common to addan activator of protein kinase C such as12-O-tetradecanoylphorbol-13-acetate (TPA) to activate a processingenzyme (ADAM12 in the case of HT1080).

It is possible to confirm progression of processing, that is, productionof remaining fragments or tagged remaining fragments by identifying theresultant solubilized growth factor.

As a method for identifying production of a solubilized growth factor,the following method can be used, for example, in the case of HB-EGF.

(Method of Using a Transfectant of Alkaline Phosphatase (AP))

Production of solubilized HB-EGF can be confirmed by determining the APactivity according to the method described in a PCT publication(WO01/70269).

(Precipitation Method Using Heparin-beads)

NaCl is added to 1 ml of a culture supernatant such that theconcentration of NaCl is 0.35 mols. Then, 20 μl of heparin-sepharose(heparin-beads) is added thereto, and incubated at room temperature for30 min. Then, the heparin-beads are washed with PBS three times,suspended in 0.5 ml of PBS, reacted with a HB-EGF antibody #H6 (antibodyagainst soluble HB-EGF), and reacted with AP-binding secondary antibody,and thus the AP activity is determined.

(Sandwich ELISA Method Using Diphteria Toxin and HB-EGF Antibody #H6)

Diphteria toxin (manufactured by Sigma) is turned into a solid phasewith 3 μg/ml of PBS in 100 μl /well in a 96-well immunoplate. A cellculture supernatant is added in an amount of 100 μl thereto and reactedat room temperature for 2 hours, followed by reaction with biotin-taggedprimary antibody, and AP-tagged streptavidin, and thus the AP activityis detected. The plate is washed with PBS/Tween (0.05%) after everyreaction.

It should be noted that for processing, endocerine-1 or the like can beused in place of TPA.

The processing process can be varied with the test cell, but generallytakes 30 to 90 min in the case of using TPA and takes about one to twohours in the case of using endocerine-1.

After the processing process ends, the procedure goes to the processesfor detecting or quantitatively determining of the taggedtranscriptional regulator.

Hereinafter, the processes for detecting or quantitatively determiningof the tagged transcriptional regulator will be described by taking thePLZF protein as an example.

The PLZF protein can be tagged with a fluorescent protein such as ayellow fluorescent protein (YFP), a cyan fluorescent protein (CFP), anda green fluorescent protein (GFP) e.g., the proteins sold under thetrademark LIVING COLORS by Clontech of California, USA) as a fluorescenttag. Alternatively, a highly antigenic synthetic peptide such assynthetic octapeptide (e.g., the peptide sold under the trademark FLAGby Sigma-Aldrich Co. of Missouri, USA) is used as a tag for the PLZFprotein, and the tag is detected using, for example, an antibody towhich a fluorescent tag such as a fluorescent protein or a coloringmatter is bound.

The nuclear export of the PLZF protein is evaluated after the processingprocess ended and a predetermined time has passed.

More specifically, a tagged protein (CFP-PLZF) obtained by binding, forexample, a cyan fluorescent protein (CFP) to the N terminal of the PLZFprotein is internalized in a plasmid, and the plasmid is transfectedinto a test cell by an ordinary method. Then, after a processingprocess, the extent of the nuclear export is evaluated with afluorescent microscope (e.g.,a microscope sold under the trademarksECLIPSE TE300, ZEISS LSM 510 or the like by Nikon Corporation of Japan).

The extent of the nuclear export can be determined by, for example,detecting or quantitatively determining either the taggedtranscriptional regulators that remain in the nucleus or the taggedtranscriptional regulators that are transported from the nucleus. Forexample, in the case of detecting or quantitatively determining thetagged transcriptional regulators that remain in the nucleus, the numberof the cells in which the nuclear export of the tagged PLZF protein doesnot occur is counted, and the ratio thereof to the number of thetransfectant cells is calculated. Alternatively, the inhibitory effecton the nuclear exports of the PLZF protein of a substance to be testedis evaluated by measuring the fluorescence intensity derived from thetagged PLZF protein in the nucleus and comparing the same with that ofthe control transfectants.

The method for determining the extent of the nuclear export referred toin the present invention includes a method for determining the extend ofthe translocation of the tagged transcriptional regulator to thevicinity of the nuclear membrane, which is a pre-stage of the nuclearexport.

Furthermore, as described above, it is observed that in the celltransfected with HB-EGF that is modified with a fluorescence in the Cterminal in the intracellular domain, when the extracellular domain ofthe HB-EGF is subjected to processing by a solubilizing enzyme, thetagged substances accumulate around the nucleus in the cell after havingapproached a golgi apparatus.

Therefore, in the present invention, another method for determining theeffect of inhibiting intracellular signaling includes the step ofdetermining the effect of inhibiting signaling from a remaining fragmentto an intranuclear transcriptional regulator, instead of the step ofdetermining the extent of the nuclear export of a tagged transcriptionalregulator.

Specific examples of the signaling from a remaining fragment to anintranuclear transcriptional regulator includes approach of a remainingfragment to a golgi apparatus, separation afterwards, accumulationaround the nucleus, transport into the nucleus, or binding or approachto an intranuclear transcriptional regulator, interaction with anintranuclear transcriptional regulator, formation of a complex of aremaining fragment, an intranuclear transcriptional regulator and DNA,and the like.

The inventors of the present invention clarified that when thetranscriptional regulator is a protein having BTB/C₂H₂Zn finger motifs,the approach of the remaining fragment to the intranucleartranscriptional regulator or interaction therebetween occurs between theremaining fragment and the Zn finger region of the protein.

Therefore, one form of the signaling from a remaining fragment to anintranuclear transcriptional regulator in the screening method of thepresent invention is signaling mediated by the Zn finger region of anintranuclear transcriptional regulator.

Therefore, the method for producing a cytostatic agent of the presentinvention can be achieved by using a substance bound to the Zn fingerregion of a protein having BTB/C₂H₂Zn finger motifs, or a substancebound to a binding region to a protein having BTB/C₂H₂Zn finger motifsin the remaining fragment of a cell membrane-anchored growth factor asan active component. For example, an antibody against the Zn fingerregion or an antibody against the remaining fragment of a cellmembrane-anchored growth factor can be used as active components for theproduction.

The Zn finger region refers to a region including C₂H₂Zn finger motifs.More specifically, it refers to a region including the following 1) and2).

-   1) Two C₂H₂Zn finger motifs-   2) A region having a TGEKPY (SEQ ID NO:2) sequence that couples the    two finger motifs described in 1)

More specifically, when the protein having BTB/C₂H₂Zn finger motifs is aPLZF protein, the remaining fragment is bound to Zn6 and Zn7, which arethe sixth and the seventh finger motifs, respectively, of nine Zn fingermotifs and the TGEKPY (SEQ ID NO:2) sequence that is sandwichedtherebetween. However, only the region of Zn6 to Zn7 is insufficient forbinding, and the region of Zn5 to Zn8 is necessary, as shown in TestExample 1 below.

When the protein having BTB/C₂H₂Zn finger motifs is a Bcl-6 protein, theremaining fragment is bound to Zn4 and Zn5, which are the fourth and thefifth finger motifs, respectively, of six Zn finger motifs and theTGEKPYP (SEQ ID NO:3) sequence (Zn4 to Zn5) that is sandwichedtherebetween.

When the protein having BTB/C₂H₂Zn finger motifs is a myoneurin protein,the remaining fragment is bound to Zn5 and Zn6, which are the fifth andthe sixth finger motifs, respectively, of eight Zn finger motifs and theTGEKPY (SEQ ID NO:2) sequence (Zn5 to Zn6) that is sandwichedtherebetween.

The tendency of the accumulation around the nucleus or the nuclearimport of the tagged substance can be evaluated in the same manner asdescribed above, that is, by counting the number of transfectant cellsin which the accumulation does not occur, or measuring the fluorescenceintensity derived from the tagged substance around or in the cell.

When a protein that is a remaining fragment artificially produced byanother method than processing is used as a protein for screening acytostatic agent, an NLS (nuclear localization signal) protein as amembrane-anchored factor can be bound to the fragment. Therefore, a geneencoding the amino acid sequence of this protein for screening is usefulfor producing this protein that is can be used for screening kit.

The site to be tagged NLS in this case is not necessarily an amino acidin the C terminal, but can be any site, as long as the signaling is notaffected.

The cytostatic agent selected by the screening method of the presentinvention is a substance that inhibits signaling in the cell mediated bythe remaining fragment of a cell membrane-anchored growth factor, andexamples of the candidates can include the following:

(I) One of the candidates is a substance that acts on a regioninteracting with a remaining fragment (b) in a transcriptional regulator(a).

More specifically, the substance can be a protein that has the same or asimilar structure to the remaining fragment and that antagonizes theoriginal remaining fragment and inhibits the signaling of the remainingfragment.

When this protein having a structure similar to that of the remainingfragment is used as a cytostatic agent, in order to increase theantagonist properties against the original remaining fragment, it ispreferable to transfect the protein into a cell in a higherconcentration than that of an endogenous cell membrane-anchored growthfactor.

In addition, for example, the following substance can be used, and itcan be used as a single substance or in the form of an aggregate of aplurality of substances.

When the transcriptional regulator (a) is a protein having BTB/C₂H₂Znfinger motifs, a substance inhibiting intracellular signaling mediatedby the remaining fragment (b) is a substance that is bound to orapproaches the Zn finger region of the protein, which is a region thatinteracts with (b) in (a), and inhibits the interaction between the Znfinger region and the remaining fragment.

More specifically, it is a substance that is bound to or approaches atleast one site of the two motifs described in 1) and the regiondescribed in 2), but preferably is bound to or approaches two sites.This binding or approach is not necessarily throughout each region, butcan be in such an extent that the remaining fragment can besubstantially inhibited from being bound to or approaching the region.

-   1) Two C₂H₂Zn finger motifs-   2) A region having a TGEKPY (SEQ ID NO:2) sequence that couples the    two finger motifs described in 1)

When the protein having BTB/C₂H₂Zn finger motifs is a PLZF protein, thetwo C₂H₂Zn finger motifs referred to in 1) correspond to at least two ofthe fifth to the eighth Zn finger motifs, that is, Zn5 to Zn8, of nineZn finger motifs. The region referred to in 2) corresponds to the TGEKPY(SEQ ID NO:2) sequence between Zn6 and Zn7 (Test Example 1).

When the protein having BTB/C₂H₂Zn finger motifs is a Bcl-6 protein, thetwo C₂H₂Zn finger motifs referred to in 1) correspond to the fourth andthe fifth Zn finger motifs, that is, Zn4 and Zn5, of six Zn fingermotifs. The region referred to in 2) corresponds to the TGEKPYP (SEQ IDNO:3) sequence (Test Example 2).

When the protein having BTB/C₂H₂Zn finger motifs is a myoneurin protein,the two C₂H₂Zn finger motifs referred to in 1) correspond to the fifthand the sixth Zn finger motifs, that is, Zn5 and Zn6, of the eight Znfinger motifs. The region referred to in 2) corresponds to the TGEKPY(SEQ ID NO:2) sequence.

As the substance that is bound to or approaches at least one site of theregions including 1) and 2), pseudo-peptide or a pseudo-syntheticcompound of the remaining fragment of the EGF family protein, orantibodies against the region including 1) and 2) can be used.

The antibodies against the region including 1) and 2) can be produced byan ordinary method in which mice or the like are immunized, using atleast one site of the two motifs described in 1) and the regiondescribed in 2) as an antigen. However, in order to inhibit signalingmore reliably, a combination of antibodies against two respective sitesas an antigen or an antibody that can recognize the two sites at onetime is preferable.

(II) Another substance that inhibits intracellular signaling mediated bythe remaining fragment is a substance that acts on a region interactingwith (a) in (b).

More specifically, the substance can be an antibody against theremaining fragment (b) or a protein having the same or a similarstructure to the intranuclear transcriptional regulator (a), and thesesubstances antagonize the original intracellular transcriptionalregulator (a) and inhibit the signaling into the intracellulartranscriptional regulator.

When this protein having a structure similar to that of theintracellular transcriptional regulator (a) is used as a cytostaticagent, in order to increase the antagonist properties against theoriginal intracellular transcriptional regulator (a), it is preferableto transfect the protein into a cell in a higher concentration than thatof an endogenous intracellular transcriptional regulator (a).

When the transcriptional regulator (a) is a protein having BTB/C₂H₂Znfinger motifs, a substance inhibiting intracellular signaling mediatedby the remaining fragment (b) is a substance that acts on a regioninteracting with the Zn finger region of the protein, which is a regionthat interacts with (a) in (b), and inhibits the interaction between theZn finger region and the remaining fragment.

More specifically, it is a substance that antagonizes the intranucleartranscriptional regulator (a) and acts on a region in (b) with which atleast one site of the two motifs described in 1) and the regiondescribed in 2) in (a) interacts. An antagonizing substance thatantagonizes the intranuclear transcriptional regulator (a) in at leasttwo sites is preferable. This interaction is not necessarily performedthroughout each region of 1) and 2) in (b), but can be in such an extentthat the intranuclear transcriptional regulator (a) can be substantiallyinhibited from interacting with the region.

-   1) Two C₂H₂Zn finger motifs-   2) A region having a TGEKPY (SEQ ID NO:2) sequence that couples the    two finger motifs described in 1)

As the substance that interacts with at least one site of the regionsincluding 1) and 2), pseudo-peptide or a pseudo-synthetic compound of atleast a portion of the Zn finger region in the intracellular regulator,or antibodies against the region that interacts with the Zn fingerregion of the intranuclear regulator in the remaining fragment can beused.

These antibodies also can be produced by an ordinary method in whichmice or the like are immunized, using at least a portion of the regionthat interacts with the Zn finger region of the intranuclear regulator(a) in the remaining fragment as an antigen. However, in order toinhibit signaling more reliably, it is preferable to use an antibodythat can recognize as many regions as possible of the regions thatinteracts with the Zn finger region.

The inventors of the present invention confirmed that the nuclear exportof PLZF is inhibited by a “rabbit polyclonal antibody using a remainingfragment of HB-EGF as an antigen”.

When the remaining fragment (b) is a remaining fragment of HB-EGF, anantibody against at least a portion of the sequence of 14 amino acidsfrom the 185^(th) to 198^(th) amino acids (SEQ ID NO:4) from the aminoterminal of HB-EGF before processing, which is a region that interactswith the intranuclear regulator (a), can be used.

These antibodies also can be produced by an ordinary method in whichmice or the like are immunized, using at least a portion of the sequenceof 14 amino acids from the 185^(th) to 198^(th) amino acids (SEQ IDNO:4) as an antigen. However, in order to inhibit signaling morereliably, it is preferable to use an antibody against as many amino acidportions as possible of the amino acid sequence from the 185^(th) to198^(th) amino acids (SEQ ID NO:4).

Another example of the substance inhibiting intracellular signalingmediated by the remaining fragment of a cell membrane-anchored growthfactor can be a substance inhibiting the intranuclear transcriptionalregulator from being transported from the nuclear. Specific examplesthereof include a substance inhibiting interaction between theintranuclear transcriptional regulator and exportin such as reptomycinB, which inhibits the binding or approach of a PLZF protein to CRM1 orthe like, which is an exportin involved in the nuclear export thereof.

These cytostatic agents of the present invention can be used to treatdiseases caused by cell growth.

TEST EXAMPLE 1 Binding Region of HB-EGF Remaining Fragment with Respectto PLZF Protein

1. Test Method

(1) In order to specify a region of PLZF protein that interacts with aremaining fragment, GST pull-down assay was performed.

(2) GST, GST binding HB-LGF remaining fragments that were expressed andisolated with E. coli BL21 strain according to the GST pull-down assaystandard protocol were used. The fragments were bound to GlutathioneSepharose beads, and then cell lysates containing various FLAG-taggedPLZF protein derivatives were incubated with 20 .mu.l of the beads at4.degree. C. for two hours. After washing with water, the bound proteinwas analyzed by immunoblotting using anti-FLAG antibody (referring tothe peptide sold under the trademark FLAG by Sigma Aldrich Co.).2. Test Results

The remaining fragments of HB-EGF were bound to the following PLZFprotein derivatives as firmly as the wild-type PLZF.

-   1) Derivatives consisting only of a region of Zn1 to Zn9 of the PLZF    protein-   2) Derivatives consisting only of a region of Zn4 to Zn9 of the PLZF    protein-   3) Derivatives consisting only of a region of Zn5 to Zn8 of the PLZF    protein

The remaining fragments of HB-EGF were bound to the following PLZFprotein derivatives, although not so firmly as the wild-type PLZF.

-   4) Derivatives consisting only of a region of Zn1 to Zn6 of the PLZF    protein

On the other hand, the remaining fragments of HB-EGF were not bound tothe following PLZF protein derivatives.

-   5) Derivatives with deletion of the Zn finger region of the PLZF    protein-   6) Derivatives consisting only of the BTB region of the PLZF protein-   7) Derivatives consisting only of a region of Zn6 to Zn7 of the PLZF    protein-   8) Derivatives with deletion of a region of Zn6 to Zn7 of the PLZF    protein

The results above indicate that the region of Zn5 to Zn8 covering theregion Zn6 to Zn7 is necessary.

TEST EXAMPLE 2 Binding Region of HB-EGF Remaining Fragment with Respectto Bcl-6 Protein

1. Test Method

The binding region of HB-EGF remaining fragments with respect to Bcl-6protein was investigated in the same method as in Test Example 1

2. Test Results

The remaining fragments of HB-EGF were bound firmly to the followingBcl-6 protein derivatives.

-   1) Derivatives consisting only of a region of Zn1 to Zn6 of the    Bcl-6 protein-   2) Derivatives consisting only of a region of Zn1 to Zn5 of the    Bcl-6 protein-   3) Derivatives consisting only of a region of Zn2 to Zn6 of the    Bcl-6 protein-   4) Derivatives consisting only of a region of Zn3 to Zn6 of the    Bcl-6 protein-   5) Derivatives consisting only of a region of Zn4 to Zn6 of the    Bcl-6 protein

The remaining fragments of HB-EGF were bound to the following Bcl-6protein derivatives, although not so firmly as the above derivatives.

-   6) Derivatives consisting of Zn1 to Zn4 and a TGEKPY (SEQ ID NO:2)    sequence bound to the C terminal of Zn4 of the Bcl-6 protein-   7) Derivatives consisting of Zn5 to Zn6 and a TGEKPYP (SEQ ID NO:2)    sequence bound to the N terminal of Zn5 of the Bcl-6 protein

The remaining fragments of HB-EGF were not bound to the following Bcl-6protein derivatives.

-   8) Derivatives consisting only of a region of Zn1 to Zn4 of the    Bcl-6 protein-   9) Derivatives with delection of a region of Zn1 to Zn3 of the Bcl-6    protein-   10) Derivatives with delection of a region of Zn1 to Zn2 of the    Bcl-6 protein-   11) Derivatives with delection of a region of Zn5 to Zn6 of the    Bcl-6 protein

The results above indicate that the TGEKPY(P) (SEQ ID NO:2 or SEQ IDNO:3) sequence between Zn4 and Zn5 and at least one region of Zn4 andZn5 (preferably both) are necessary.

TEST EXAMPLE 3 Nuclear Export of PLZF Protein by Processing

1. Test Method

The following cell groups were used:

-   1) Human fibrosarcoma HT1080 cells-   2) HT1080 cells to which HB-EGF has been transfected (HT1080/HB-EGF)-   3) Metalloprotease domain-deleted mutant of ADAM12 in the above    cells (HT1080/ΔMP-ADAM12/HB-EGF)-   4) HT1080 cells to which processing resistant HB-EGF has been    transfected (HT1080/HB-EGF-UC)

1) to 3) already have been reported (Asakura et al., Nat. Med., 8, 35-40(2002))

44) was produced by transfecting an expression vector of processingresistant (uncleavable) HB-LGF (Hirata et al., BBRC, 283, 915-922(2001)) into HT1080 cells using LIPOFECTAMTNE 2000 reagent (sold byInvitrogen of California, USA).

The PLZF protein was tagged by transfecting cDNA of a cyan fluorescentprotein (CFP) into the N-terminal side of CDNA of the PLZF protein, andthe tagged CDNA of the PLZF protein was transfected into each cellutilizing a commercially available vector (pECFP-C1; Clontech).

Each transfectant containing CFP-PLZF was cultured for 24 hours, andthen 100 nM TPA was added thereto for treatment for 60 min. The number(N) of the cells expressing CFP-PLZF in the nucleus and the number (C)of the cells in which CFP-PLZF has leaked into the cytoplasm are countedunder observation with a fluorescent microscope before and after the TPAtreatment, and the ratio of the number (N) of the cells expressingCFP-PLZF in the nucleus to the total number (N+C) of the cellsexpressing CFP-PLZF is calculated and thus obtained (N/(N+C)×100 (%)).The wild-type HT1080 cells (the above described 1)) were used as thecontrol for comparison.

2. Test Results

FIG. 1 shows the results. The TPA treatment triggered processing, andthe remaining fragment of HB-EGF caused the nuclear export of the PLZFprotein present in the nucleus of the cell. As a result, inHT1080/HB-EGF, the TPA treatment reduced the fluorescence intensity ofCFP-PLZF present in the nucleus, whereas in the control cells (HT1080)in which HB-EGF is not present, the fluorescence intensity of CFP-PLZFwas not changed between before and after the TPA treatment. On the otherhand, in HT1080/ΔMP-ADAM12/HB-EGF, the TPA treatment did not triggerprocessing. In HT1080/HB-EGF-UC, since HB-EGF is resistant againstprocessing, the fluorescence intensity of CFP-PLZF also was not changedin these cells between before and after the TPA treatment.

TEST EXAMPLE 4 Expression of Cyclin A in Response to the Nuclear Exportof PLZF Protein

1. Test Method

Expression of cyclin A in response to the nuclear export of PLZF proteinwas traced by immunofluorescence. cDNA of a synthetic octapeptide (soldunder the trademark FLAG by Sigma-Aldrich Co. of Missouri, USA) havinghigh antigenicity was bound to the N terminal of the cDNA of the PLZFprotein, and the cDNA of the PLZF protein was transfected intoHT1080/HB-EGF using a commercially available vector (pcDNA3. 1/Hygro(+);Invitrogen).

The cultured transfectant was subjected to a TPA treatment (100 nM, 8hours), and then washed with phosphate-buffered saline (PBS) andimmobilized with 4% paraformaldehyde (PFA)/PBS (4° C., 10 min).Subsequently, the transfectant was treated with 0.5% triton X-100/PBSand 1% bovine serum albumin (BSA).

A mouse monoclonal IgG antibody against the transfected octapeptide anda rabbit polyclonal antibody against cyclin A were added, and incubatedfor one hour and the resultant was washed with PBS. Then,rhodamine-conjugated goat anti-mouse IgG antibody and afluorescence-tagged (trademark: Alexa Fluor 488, Molecular Probes Co.)goat anti-rabbit IgG antibody were added thereto, followed by furtherone hour incubation. The resultant was washed with PBS, and thereafterthe cells were mounted with PBS containing 5000 glycerol and 0.1%phenylene diamine and observed with a fluorescence microscope, and thenthe fluorescence intensity was measured with image processing softwaresold under the trademark LUMINA VISION by Mitani Corporation of Japan).

2. Test Results

Regarding the nucleus of each cell used in the test, the differencebetween the presence and the absence of the TPA treatment was evaluatedwith the vertical axis showing the fluorescence intensity derived fromoctapeptide bound to the PLZF protein, and the horizontal axis showingthe fluorescence intensity derived from cyclin A (FIG. 2). It wasobserved that the expression of cyclin A in the nucleus that had beensuppressed by the transfection of the octapeptide-tagged PLZF proteinwas liberated from the suppression, as a result of the nuclear export ofthe PLZF protein due to the TPA treatment, and the fluorescenceintensity of cyclin A converged in a high intensity region (in thevicinity of 0.8).

Such an effect was not seen in HT1080/ΔMP-ADAM12/HB-EGF orHT1080/HB-EGF-UC.

TEST EXAMPLE 5 Change in the Amount of BrdU Internalized by Processing

1. Test Method

A synthetic octapeptide-tagged PLZF protein (see Test Example 4) wastransfected to each of the cell groups (HT1080, HT1080/HB-EGF,HT1080/ΔMP-ADAM12/HB-EGF and HT1080/HB-EGF-UC) of Test Example 3.

Each transfectant was cultured in an eagle MEM medium (containing 3%FCS) for 36 hours, and incubated in the same medium (containing 10% FCS)for 12 hours. Thereafter, 100 nM TPA was added thereto and a treatmentwas performed for 2 hours. Then, 3 μg/ml of BrdU was added to themedium, followed by further two hours incubation. After immobilizationwith 4% PFA/PBS and permeation with 0.5% tritonX-100/PBS, the cells weretreated with 2N hydrochloric acid for 30 min and subsequently with 1%BSA for 30 min.

A mouse monoclonal IgG antibody (Shigma) against the octapeptide portionand a rat monoclonal IgG antibody (Abeam Ltd.) against BrdU were added,and the resultant was incubated for one hour and washed with PBS. Then,isothiocyanate-conjugated goat anti-mouse IgG antibody and arhodamine-conjugated goat anti-rat IgG antibody (sold by CHEMICONinternational Inc. of California, USA) were added thereto, followed byfurther incubation. The resultant was washed with PBS, and thereafterthe cells were mounted with PBS containing 50% glycerol and 0.1%phenylene diamine and observed with a fluorescence microscope.

Regarding the octapeptide-tagged PLZF protein transfectants andnon-transfectants, the ratios in the number of cells internalizing BrdUwere compared in the presence and the absence of the TPA treatment.

2. Test Results

FIG. 3 shows the results. The internalization ratio of BrdU in thenon-transfectants of the PLZF protein is generally about 40 to 50%, andthere was no change with the TPA treatment. However, in PLZFtransfectants, the action of cyclin A was suppressed by the suppressoreffect of the expressed PLZF protein, and the cell cycle did not proceedfurther than the S-phase (DNA synthetic-phase), and thus theinternalization ratio of BrdU was increased. In this case, when the TPAtreatment was performed, the nuclear export of the PLZF protein wascaused, so that the suppressor effect of this protein was weakened.Thus, cyclin A was activated and the cell cycle proceeded further ontothe G1-phase from the S-phase. Consequently, the internalization ratioof BrdU decreased. However, in HT 1080 in which almost no HB-EGF waspresent, HT1080/ΔMP-ADAM12/HB-EGF in which processing is not caused, andHT1080/HB-EGF-UC in which processing is resisted, the TPA treatment didnot change the internalization ratio of BrdU.

TEST EXAMPLE 6 Nuclear Export of Bcl-6 Protein or Myoneurin Protein byProcessing

1. Test Method

Whether or not the nuclear export of transcriptional regulators occurredwas examined in the same manner as in Test Example 3, except that thefollowing cell groups were used, and Bcl-6 protein or myoneurin proteinwere used as the tagged transcriptional regulators, instead of the PLZFprotein.

-   -   1) Human fibrosarcoma HT1080 cells    -   2) HT1080 cells to which HB-EGF has been transfected        (HT1080/HB-EGF)        2. Test Results

FIGS. 4 and 5 show the results. Processing was triggered by the TPAtreatment, and the remaining fragment of HB-EGF caused the nuclearexport of the Bcl-6 protein or the myoneurin protein present in thenucleus in a cell. As a result, in the HT1080/HB-EGF, the fluorescenceintensity of CFP-Bcl-6 or CFP-myoneurin present in the nucleus wasdecreased by the TPA treatment.

TEST EXAMPLE 7 Nuclear Export of KIAA0441 Protein by Processing

1. Test Method

Whether or not the nuclear export of transcriptional regulators occurredwas examined in the same manner as in Test Example 3, except that thefollowing cell groups were used, KIAA0441 protein was used as the taggedtranscriptional regulator, instead of the PLZF protein, and epiregulin(ER) was used, instead of HB-EGF.

-   1) Human fibrosarcoma HT1080 cells-   2) HT1080 cells to which epiregulin has been transfected (HT1080/ER)    2. Test Results

FIG. 6 shows the results. Processing was triggered by the TPA treatment,and the remaining fragment of epiregulin caused the nuclear export ofKIAA0441 protein present in the nucleus in a cell. As a result, in theHT1080/ER, the fluorescence intensity of CFP-KIAA0441 present in thenucleus was decreased by the TPA treatment.

EXAMPLE

Hereinafter, the present invention will be described more specificallyby way of an example.

Inhibitory Effect of Reptomycin B Against the Nuclear Export of the PLZFProtein

-   1. A compound to be tested reptomycin B (LMB)-   2. Test method

HT1080/HB-EGF to which CFP-PLZF had been transfected (see Test Example3) was cultured for 24 hours, and then treated with 100 nM TPA in thecopresence of LMB (10 ng/ml) for 60 min. The number of the cellsexpressing CFP-PLZF in the nucleus was measured with a microscope beforeand after the TPA treatment and the ratio was calculated.

As the control, the ratio of the number of the cells expressing CFP-PLZFbefore and after the TPA treatment when the TPA treatment was performedwithout adding LMB was obtained, and compared with the ratio when LMBwas added. Thus, the compound LMB to be tested was evaluated.

-   3. Test Results

FIG. 7 shows the numbers of the cells expressing CFP-PLZF in the nucleusbefore and after the TPA treatment with and without LMB. It wasconfirmed that LMB inhibited the nuclear export of the PLZF protein.

INDUSTRIAL APPLICABILITY

When in cells transfected with amino-terminally (N terminally)fluorescence-tagged CFP-PLZF, the extracellular domain of HB-EGF wassubjected to processing by a solubilizing enzyme, the nuclear export ofthe tagged PLZF protein was observed, and this effect was suppressed bya processing inhibitor (Test Example 3).

Next, it was confirmed that PLZF proteins and cyclin A were confirmed tobe present in the nucleus by immunofluorescence, and thereafter when theextracellular domain of HB-EGF was subjected to processing by asolubilizing enzyme, the nuclear export of the PLZF protein occurred andthe fluorescence intensity of cyclin A was increased (Test Example 4).

When the PLZF protein is forcefully expressed in cells, the action ofcyclin A is suppressed and the cells concentrate on the S-phase of thecell cycle, so that the amount of internalized bromodeoxyuridine (BrdU)temporarily increases. At this point, when the extracellular domain issubjected to processing by a solubilizing enzyme, the action of cyclin Ais accelerated by the nuclear export of the PLZF protein, and the cellgrowth proceeds from the S-phase to the G2-phase, and thus the amount ofinternalized BrdU decreases (Test Example 5).

It was confirmed that even after the processing of the extracellulardomain by a solubilizing enzyme occurred, the addition of reptomycin Bactually inhibits the nuclear export of the PLZF protein (see theexample). Since reptomycin B is known as an inhibitor of nuclear export(Kudo et al., Proc. Natl. Acad. Sci. USA, 96,9112-9117(1999)) and anantitumor antibiotic, the essence of the antitumor action is to inhibitthe nuclear export of intranuclear transcriptional regulator byintracellular domain, and thus it was confirmed that the presentinvention is useful as a method for screening a cytostatic agent.

Therefore, the screening method of the present invention can beperformed as primary screening for a cytostatic agent that can inhibitthe intracellular signaling process and also can be performed assecondary screening (confirmation test) to confirm the cytostaticability (antitumor action) of a substance to be tested. Furthermore, thecytostatic agent obtained by the production method of the presentinvention is very useful as an antitumor agent, an antirheumatic agent,an agent for treating cardiac disorder, etc. that are based on a novelaction mechanism.

1. A method for screening a cytostatic agent comprising the steps of: producing within a cell at least one of (A) a remaining fragment of HB-EGF, and (B) a modified remaining fragment of HB-EGF; determining at least one of (i) an extent of export from the nucleus of the cell of a tagged-PLZF protein to which any of (A) and (B) is bound, and (ii) an extent of signalling from the remaining fragment to a PLZF protein, wherein the production and determination steps are conducted for each of the cases in the presence and in the absence of a candidate substance to be tested; correlating at least one of (i) a reduced extent of the export from the nucleus and (ii) an inhibited signalling when the candidate substance is present, as compared to when it is absent, with a cytostatic property of the candidate substance, wherein the modified remaining fragment (B) is a remaining fragment of HB-EGF in which ten of the amino acids at the C-terminal of HB-EGF have been deleted.
 2. The method for screening a cytostatic agent according to claim 1, wherein the production step is a step of producing a remaining fragment of HB-EGF by processing of the growth factor; and the determination step is a step of determining an extent of export from the nucleus of a tagged PLZF protein.
 3. The method for screening a cytostatic agent according to claim 1, wherein the production step is a step of producing a tagged remaining fragment of HB-EGF by processing of the growth factor; and the determination step is a step of determining an effect of inhibiting signaling from the tagged remaining fragment to a PLZF protein.
 4. The method for screening a cytostatic agent according to claim 3, wherein the signaling from the tagged remaining fragment to a PLZF protein in the determination step is signaling mediated by a Zn finger region of the PLZF protein.
 5. The method for screening a cytostatic agent according to claim 4, wherein the Zn finger region is a region having Zn5 to Zn8 region of a PLZF protein.
 6. The method for screening a cytostatic agent according to any one of claims 3 to 5, wherein the signaling from the tagged remaining fragment to the PLZF protein in the determination step is signaling mediated by the following portion (iv) of the tagged remaining fragment, wherein (iv) is an amino acid sequence portion corresponding to the 185^(th) to 198^(th) amino acids (SEQ ID NO:4) from an amino terminal in the HB-EGF before processing.
 7. The method for screening a cytostatic agent according to claim 2 or 3, wherein production of the remaining fragment or the tagged remaining fragment in the production step is confirmed by detection of the solubilized HB-EGF.
 8. The method for screening a cytostatic agent according to any one of claims 2 to 5, wherein at least one of a fluorescent tag and a peptide having antigenicity is used to tag or label the PLZF protein.
 9. The method for screening a cytostatic agent according to claim 8, wherein a fluorescent protein selected from the group consisting of a cyan fluorescent protein (CFP), a yellow fluorescent protein (YFP), and a green fluorescent protein (GFP) is used to tag or label the PLZF protein.
 10. A method for screening a cytostatic agent comprising the steps of: producing within a cell (a remaining fragment of HB-LGF; determining at least one of (i) an extent of export from the nucleus of the cell of a tagged-PLZF protein to which the remaining fragment of HB-LGF is bound, and (ii) an extent of signalling from the remaining fragment to a PLZF protein, wherein the production and determination steps are conducted for each of the cases in the presence and in the absence of a candidate substance to be tested; correlating at least one of U) a reduced extent of the export from the nucleus and (ii) an inhibited signalling when the candidate substance is present, as compared to when it is absent, with a cytostatic property of the candidate substance. 